Innate dysfunction promotes linear growth failure in pediatric Crohn's disease and growth hormone resistance in murine ileitis

Abstract

Background: Growth failure remains a common complication of pediatric Crohn's disease (CD) and has been associated with small bowel involvement and need for surgery. We have reported that patients with elevated (≥ 1.6 μg/mL) granulocyte macrophage colony stimulating factor autoantibodies (GM-CSF Ab) are more likely to experience complicated ileal disease requiring surgery. We hypothesized that concurrent GM-CSF Ab and CARD15 risk allele carriage (C15(+) GMAb(+) ) would be associated with growth failure in CD and growth hormone (GH) resistance in murine ileitis.

Methods: We enrolled 229 pediatric CD patients at two sites and determined CARD15 genotype, serum GM-CSF Ab, and GH binding protein (GHBP), and height (HTz) and weight (WTz) z-scores at diagnosis. Ileitis was induced in card15-deficient mice by GM-CSF neutralization and nonsteroidal antiinflammatory drug (NSAID) exposure. Hepatic GH receptor (GHR) abundance and GH-dependent Stat5 activation were determined by western blot and Igf-I mRNA expression by real-time polymerase chain reaction (PCR).

Results: Mean (95% confidence interval [CI]) HTz at diagnosis was reduced to -0.48 (-4.2, 2.3) in C15(+) GMAb(+) patients, compared to -0.07 (-4.9, 3.4) in disease controls (P ≤ 0.05). Circulating GHBP, as a marker for tissue GHR abundance, was reduced in C15(+) GMAb(+) patients. Hepatic GHR abundance, GH induction of Stat5 tyrosine phosphorylation, and Igf-I mRNA expression were reduced in male card15-deficient mice with ileitis due to GM-CSF neutralization and NSAID exposure.

Conclusions: Innate dysfunction due to concurrent genetic variation in CARD15 and neutralizing GM-CSF Ab is associated with linear growth failure in pediatric CD, and hepatic GH resistance in murine ileitis.

Figure 1. Serum Growth Hormone Binding Protein is Reduced in CD Patients with Combined CARD15 SNP and Elevated GM-CSF Ab

Serum GHBP concentration was determined by ELISA in (A) male C15⁺GMAb⁺ CD patients (n= 30) compared to male disease controls with one or neither of these factors (n=84), *p< 0.05 by Mann-Whitney test and (B) female C15⁺GMAb⁺ CD patients (n=20) compared to female disease controls with one or neither of these factors (n=60), *p< 0.05 by unpaired t-test. Data are shown as the mean ± SEM.

Figure 2. Hepatic Growth Hormone Receptor Expression is Reduced in Male Mice with Ileitis

Ileitis was induced by NSAID exposure to Card15 −/− mice following Gm-csf neutralization. Liver protein and RNA were isolated from mice with ileitis, mice pre-treated with isotype control antibody and then exposed to NSAID (NSAID) and controls. (A and B) Ghr protein expression was determined by immunoblot and (C and D) total Ghr mRNA expression was determined by real-time PCR. Signal intensity was determined by densitometry in (A) male ileitis mice (n=6) compared to male NSAID (n=4) and controls (n=6) and (B) female ileitis mice (n=7) compared to female NSAID (n=5) and controls (n=6). β-tubulin was used to confirm uniform protein loading. Data are shown as the mean ± SEM; *p<0.05.

Figure 2. Hepatic Growth Hormone Receptor Expression is Reduced in Male Mice with Ileitis

Ileitis was induced by NSAID exposure to Card15 −/− mice following Gm-csf neutralization. Liver protein and RNA were isolated from mice with ileitis, mice pre-treated with isotype control antibody and then exposed to NSAID (NSAID) and controls. (A and B) Ghr protein expression was determined by immunoblot and (C and D) total Ghr mRNA expression was determined by real-time PCR. Signal intensity was determined by densitometry in (A) male ileitis mice (n=6) compared to male NSAID (n=4) and controls (n=6) and (B) female ileitis mice (n=7) compared to female NSAID (n=5) and controls (n=6). β-tubulin was used to confirm uniform protein loading. Data are shown as the mean ± SEM; *p<0.05.

Figure 3. Growth Hormone Induced Hepatic pSTAT5 Activation is Reduced in Male Mice with Ileitis

Ileitis was induced by NSAID exposure to Card15 −/− mice following Gm-csf neutralization. Liver nuclear protein was isolated from mice with ileitis, mice pre-treated with isotype control antibody and then exposed to NSAID (NSAID) and controls 30 minutes after human GH injection (3mcg/gm, IP). Abundance of pSTAT5 was determined by immunoblot in (A) male ileitis mice (n=7), mice with NSAID exposure alone (n=5), and controls (n=6) and (B) female ileitis mice (n=5), mice with NSAID exposure alone (n=4), and controls (n=7). Signal intensity was determined by densitometry; SHPTP1 was used to confirm loading. Data are shown as the mean ± SEM; **p<0.01.

Figure 4. Circulating LBP and the Hepatic Ghr:Igf-1 Axis in Murine Ileitis

Ileitis was induced by NSAID exposure to Card15 −/− mice following Gm-csf neutralization (n=22). Serum LBP and Igf-I were determined by ELISA. Liver Ghr and Igf-I mRNA expression were determined by real-time PCR. Spearman correlations were determined as shown.